Journal: Biomolecules

Article Title: Helicobacter pylori -Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury

doi: 10.3390/biom14070772

Figure Lengend Snippet: Both Helicobacter pylori infection and reduced Na,K-ATPase activity impair intracellular junctions in cultured cells ( A – C ) and in gerbil mucosa ( D ). HGE-20 cells were grown to confluency in Transwell™ inserts, and the transepithelial electrical resistance (TEER) was measured in the presence of H. pylori ( A ) or ouabain ( B ). The TEER is expressed as a percentage of the initial value. Initial values of the TEER in HGE-20 cells ranged from 1000 to 1500 Ω×cm 2 . Paracellular permeability of confluent HGE-20 cell layers was measured in an Ussing chamber in the presence or absence of ouabain, results are expressed as the rate of FD4 flux through the cell layer ( C ). Gerbils were euthanized and stomach tissue incubated in an Ussing chamber in the presence of ouabain, and the TEER was measured at the indicated times. Results are expressed as percent of initial value ( D ). Initial values of the TEER in gerbil gastric mucosa ranged from 75 to 100 Ω×cm 2 , consistent with prior published results . For all panels, n = 3, * p < 0.05 ( t -test), mean ± SD.

Article Snippet: Primary antibodies against Na,K-ATPase α 1 subunits (1:1000; mouse, Clone:C464.6, EMD, Millipore, Temecula, CA, USA) and Na-K-ATPase β1-subunits (1:1000; mouse, clone M17-P5-F11, Affinity Bioreagents, Golden, CO, USA) were used to detect Na,K-ATPase.

Techniques: Infection, Activity Assay, Cell Culture, Permeability, Incubation

Journal: Biomolecules

Article Title: Helicobacter pylori -Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury

doi: 10.3390/biom14070772

Figure Lengend Snippet: Human gastric organoids as a model system to study the effect of Helicobacter pylori on gastric mucosa. Human gastric antrum was obtained from de-identified sleeve gastrectomy specimens. The location of the harvested sample is indicated by the circle ( A ). Human gastric organoids were cultivated from the fresh tissue samples and plated in Matrigel with appropriate growth factors, leading to the formation of 3D organoids ( B ). Basolateral expression of Na,K-ATPase was confirmed in gastric organoids by immunofluorescence of the α 1 subunit, indicated by the red color, and F-actin was used as a counterstain, indicated by the green color ( C ). Three-dimensional organoids were converted to 2D using Matrigel-coated Transwell™ inserts and were grown into a confluent monolayer. Organoids were incubated with the vehicle or H. pylori ( D , E ), and the decrease in Na,K-ATPase subunits in the presence of H. pylori was confirmed in this model system by immunofluorescence ( D ) or Western blot ( E ). Quantification is shown in the right panels. Mean ± SD, n = 3, * p < 0.05, t -test.

Article Snippet: Primary antibodies against Na,K-ATPase α 1 subunits (1:1000; mouse, Clone:C464.6, EMD, Millipore, Temecula, CA, USA) and Na-K-ATPase β1-subunits (1:1000; mouse, clone M17-P5-F11, Affinity Bioreagents, Golden, CO, USA) were used to detect Na,K-ATPase.

Techniques: Expressing, Immunofluorescence, Incubation, Western Blot

Journal: Biomolecules

Article Title: Helicobacter pylori -Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury

doi: 10.3390/biom14070772

Figure Lengend Snippet: Helicobacter pylori infection, reduced amount of Na,K-ATPase, and reduced Na,K-ATPase activity similarly decrease the TEER in human gastric organoids. ( A , B ) The TEER of 2D human gastric organoids grown on Transwell™ inserts and measured in an Endohm ® chamber was decreased after exposure to Helicobacter pylori ( A ) and in the presence of ouabain compared with the vehicle ( B ). Western blot showing the silencing of the Na,K-ATPase α 1 subunit ( C ). The TEER of 2D human gastric organoids grown on Transwell™ inserts and measured in an Endohm ® chamber was decreased after silencing the Na,K-ATPase α 1 subunit ( D ). Initial values of the TEER in 2D gastric organoids ranged from 800 to 1300 Ω×cm 2 . Mean ± SD, significant differences from control, t -test, n = 3, * p < 0.05 ( A , C , D ) and n = 5, ** p < 0.01 ( B ).

Article Snippet: Primary antibodies against Na,K-ATPase α 1 subunits (1:1000; mouse, Clone:C464.6, EMD, Millipore, Temecula, CA, USA) and Na-K-ATPase β1-subunits (1:1000; mouse, clone M17-P5-F11, Affinity Bioreagents, Golden, CO, USA) were used to detect Na,K-ATPase.

Techniques: Infection, Activity Assay, Western Blot, Control

Journal: Biomolecules

Article Title: Helicobacter pylori -Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury

doi: 10.3390/biom14070772

Figure Lengend Snippet: Reduced Na,K-ATPase activity and Helicobacter infection similarly impair adherens junctions in human gastric organoids. Immunofluorescence of 2D gastric organoids using E-cadherin antibodies showed a decreased E-cadherin signal at the cell junctions in the presence of H. pylori ( A ) or ouabain ( D ) and an increased intracellular accumulation of E-cadherin ( B , E ). E-cadherin clusters were decreased in size in the presence of H. pylori ( C ) or ouabain ( F ), suggesting injury to junctions. Mean ± SD, significant differences from “no Hp” or “no ouabain”, t -test. n = 5, ** p < 0.01, *** p < 0.001 ( B , F ).

Article Snippet: Primary antibodies against Na,K-ATPase α 1 subunits (1:1000; mouse, Clone:C464.6, EMD, Millipore, Temecula, CA, USA) and Na-K-ATPase β1-subunits (1:1000; mouse, clone M17-P5-F11, Affinity Bioreagents, Golden, CO, USA) were used to detect Na,K-ATPase.

Techniques: Activity Assay, Infection, Immunofluorescence

Journal: International Journal of Oncology

Article Title: Dual targets of lethal apoptosis and protective autophagy in liver cancer with periplocymarin elicit a limited therapeutic effect

doi: 10.3892/ijo.2023.5492

Figure Lengend Snippet: PPM-induced macroautophagy counteracted the apoptosis cell death. (A) LC3 protein expression levels of HepG2 cells with ATP1A1 knockdown and/or treated with PPM (100 nmol/l). HepG2 cells were treated with PPM (100 nmol/l) and/or 3-MA (5 mmol/l) for 24 h; (B) cell viability was then determined using MTS assays and (C) apoptosis was analyzed using flow cytometry. GAPDH or β-actin was used as a loading control. Data are presented as mean ± SD, n=3; ** P<0.01 and *** P<0.001 vs. control. ### P<0.001 vs. PPM. n.s., not significant; PPM, periplocymarin; siRNA, short interfering; LC3, microtubule-associated protein light chain 3; ATP1A1, ATPase Na + /K + transporting subunit α1.

Article Snippet: ATPase Na + /K + Transporting Subunit α 1 (ATP1A1) siRNA and its negative control siRNA (cat. No. siN0000001-1-5) were purchased from Guangzhou RiboBio Co., Ltd.

Techniques: Expressing, Knockdown, Flow Cytometry, Control